The localization of mRNA is a widespread form of gene regulation used to restrict the synthesis of specific proteins to distinct regions of cells. While the molecular mechanisms of mRNA localization are unknown, it has been shown that localized mRNAs are transported along the cytoskeleton in association with large subcellular structures, often referred to as RNA granules. The general goal of this proposal is to identify and characterize proteins responsible for RNA localization in Xenopus oocytes. In Xenopus, the maternal Vg1 mRNA is localized by a microtubule and actin dependent process to the vegetal cortex of oocytes during oogenesis. Like other localized mRNAs, the signals for Vg1 mRNA localization reside within its 3' UTR. Previously, Deshler identified an RNA binding protein, Vera, that is involved in the localization of Vg1 mRNA. He also showed that Vera specifically recognizes localization signals in the Vg1 3' UTR. An intriguing aspect of Vera is that it is associated with ER membranes suggesting that the ER serves as a transport vehicle for Vg1 mRNA. The proposed experiments seek to identify and characterize various molecular components of the Vg1 mRNA localization machinery. The specific aims are: 1) a mutational analysis of Vera to identify domains that mediate its specific binding to RNA localization signals in the Vg1 3' UTR; 2) the identification of proteins that interact with Vera and may mediate its association with the ER; and 3) a search for new RNA binding proteins that are both associated with large subcellular structures and specifically recognize RNA localization signals in Xenopus oocytes. The results of the proposed research should give insights into the molecular mechanism(s) responsible for the assembly of RNA granules and the association of these structures with the cytoskeleton.